YIC-IR  > 中科院烟台海岸带研究所知识产出
迪茨氏菌CN-3的石油烃降解机制及烷烃羟化酶功能研究
陈卫卫
Subtype博士
Thesis Advisor胡晓珂
2020-11-30
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Place of Conferral北京
Keyword石油烃 迪茨氏菌 降解 机制 烷烃羟化酶
Abstract近年来,由于自然或人为原因造成的石油污染日益严重,对生态环境和人类健康造成了长期和恶劣的影响。相比物理和化学修复,微生物修复具有经济、环保、高效、可持续等优势,成为当今非常重要的环境修复技术。微生物修复石油污染的关键是寻找优质的菌种资源,特别是降解活性高、环境适应性强、底物范围广的微生物。本研究从渤海蓬莱19-3溢油平台的海洋沉积物中,分离到一株耐盐的石油降解菌株Dietzia sp. CN-3,其具有石油烃降解活性高、底物谱范围广和环境耐受性好的优势。利用基因组测序分析和转录组测序分析,预测了烷烃降解相关的生物学过程和代谢途径,包括最为关键的两个烷烃羟化酶基因(alkB和CYP153)。通过荧光定量PCR、插入失活和异源表达等分子生物学手段鉴定了alkB和CYP153的功能。最后,将具备不同石油烃降解和表面活性剂生产能力的迪茨氏菌CN-3与不动杆菌HC8-3S进行组合,评价其石油降解性能,为石油污染的生物修复提供理论依据和技术支持。主要的研究内容和结论如下: 一、从渤海蓬莱19-3溢油平台的海洋沉积物中,分离到一株耐盐的石油烃降解菌株迪茨氏菌CN-3。该菌株能够高效地降解石油烃(91.6%)、单一的烷烃和芳香烃,包括直链烷烃(C10-C36)、支链烷烃(姥鲛烷和植烷)、环烷烃和芳香烃(菲和芘)。迪茨氏菌CN-3具有良好的环境耐受性(温度4-42℃,pH 5-10和盐度0-160 g/L),特别是在高盐度(85 g/L)条件下,石油烃降解活性(85.6%)几乎不受影响。迪茨氏菌CN-3通过生产表面活性剂或改变细胞表面疏水性,加速对不同石油烃的降解。 二、完成了迪茨氏菌CN-3的基因组测序,为探究不同的生物学过程和代谢途径提供全面的遗传背景。基因组学分析显示,迪茨氏菌CN-3的基因组中包含1个染色体基因组和2个质粒基因组,总长度为3741379 bp,GC含量为70.66%。共编码3496个基因,编码基因长度占基因组总长度的88.83%。基因组中含有50个tRNA,9个rRNA和12个基因岛。基因组中注释出大量与烷烃降解相关的基因,包括1个AlkB类型的烷烃羟化酶基因和1个细胞色素P450家族的CYP153烷烃羟化酶基因,以及烷烃单末端氧化途径中涉及到的多种酶,如乙醇脱氢酶、乙醛脱氢酶、脂酰辅酶A合成酶等。另外,基因组中注释出19个芳香族化合物降解相关的基因,如邻苯二酚1,2-双加氧酶基因catA、邻苯二酚2,3-双加氧酶基因catE等。在邻苯二酚途径中鉴定出比较完整的基因,暗示该途径在CN-3菌株降解芳香族化合物的过程中起主要作用。 三、完成了迪茨氏菌CN-3分别在琥珀酸钠和正十六烷培养下的转录组测序,全面地分析基因在不同生物学过程中的表达情况。转录组学分析显示,在正十六烷诱导下,迪茨氏菌CN-3的转录谱发生了明显的变化,共找到546个差异表达基因(筛选标准是错误发生率FDR<0.05和差异表达量|Log2FC|>1),其中168个基因表达显著上调,378个基因表达显著下调。其中,涉及到脂类代谢、氨基酸代谢、碳水化合物代谢、异生物质生物降解和代谢、转录和翻译等生物学过程中的差异表达基因较多,并发现了31个潜在的新基因。起始烷烃降解的烷烃羟化酶基因alkB、CYP153、铁氧还蛋白基因Fdx和铁氧还蛋白还原酶基因FdR强烈地上调表达(log2FC值5.3065-6.198),与荧光定量PCR验证结果几乎一致。同时,与正十六烷单末端氧化途径相关的基因也都有不同程度的上调表达,暗示CN-3菌株通过单末端氧化途径代谢正十六烷。 四、结合体内和体外实验,证实了烷烃羟化酶基因alkB和CYP153在不同烷烃降解中的功能,与前人报道不同,可能是一种潜在的新机制。通过对alkB和CYP153基因的扩增和同源性分析发现,alkB对应的氨基酸序列中具有4个保守基序Hist-1、Hist-2、Hist-3和HYG-motif,被鉴定为AlkB类型的烷烃羟化酶;CYP153基因属于细胞色素P450的CYP153A家族。利用pK18自杀质粒在迪茨氏菌中成功构建了插入失活突变株ΔalkB和ΔCYP,证明了alkB和CYP153基因在中链烷烃、支链烷烃和长链烷烃降解中均发挥作用,但对不同烷烃的利用存在偏好性。CYP153基因在中链烷烃和支链烷烃降解中具有优势,而alkB基因在长链烷烃利用中更具优势。对于超长链烷烃C28,alkB和CYP153基因具有协同作用。上述结果与不同烷烃诱导下alkB和CYP153基因的转录水平分析相一致。此外,从合成生物学的角度,以Pseudomonas putida F1为底盘细胞,人工引入alkB,初步构建了石油烃降解细胞工厂,使F1菌株获得了中链和长链烷烃(C14、C16、C24和C26)降解能力,扩大了底物谱,有潜力成为一株石油污染生物修复的工程菌。 五、选取具有不同石油降解特性和生物表面活性剂生产能力的Dietzia sp. CN-3和Acinetobacter sp. HC8-3S,构建了优势互补的复合菌,并开展了石油污染生物修复的微宇宙实验。相比于单一菌株,该复合菌能够显著地提高石油降解能力,10天内降解95.8%的石油烃,并能够在广谱的pH(4-10)和盐度(0-120 g/L)范围内高效地降解石油。在石油污染生物修复的微宇宙实验中,添加复合菌的生物强化处理组对石油的降解率达到485.8 mg kg-1 d-1,明显高于前人的报道,展示了该复合菌在石油污染生物修复方面的应用潜力。 综上所述,本论文对迪茨氏菌CN-3的石油烃降解效能和机制,以及烷烃羟化酶的功能进行了深入和详尽的分析,证实了烷烃羟化酶基因alkB和CYP153在不同烷烃降解中的分工与合作的作用机制,可能是一种潜在的新机制。本工作的完成将推进迪茨氏菌降解石油的研究,并为其他石油降解菌的相关研究奠定基础,具有重要的参考意义;同时,本工作的完成将会加快迪茨氏菌的遗传改造和环境应用,为石油污染的生物修复提供菌种资源和技术支持。
Other AbstractIn recent years, crude oil pollution caused by natural or anthropogenic factors, has become increasingly serious, leading to long-term and devastating effects on the ecological system and human health. Bioremediation, using microorganisms to degrade crude oil, has been considered as an important environmental remediation technology, which is also more economical, effective, versatile and environmentally friendly than physical and chemical remediation. Particularly, the key of crude oil pollution bioremediation is to find microbial strains with high degradation activity, strong environmental adaptability and wide substrate range. In this study, Dietzia sp. CN-3 was isolated from the marine sediments of Penglai 19-3 platform in the Bohai Sea. D. sp. CN-3 had high petroleum degradation activity, wide substrate range and strong environmental adaptability, which could be potential in crude oil pollution bioremediation. Due to genomics and transcriptomics, the biological processes and metabolic pathways related to alkane degradation were predicted, especially to the alkane hydroxylase gene (alkB and CYP153). Furthermore, the function of alkB and CYP153 was identified through RT-qPCR, gene disruption and heterologous expression methods. Two bacterial strains of D. sp. CN-3 and Acinetobacter sp. HC8-3S, with sufficient laboratory research foundation, were functionally combined to construct a bacterial consortium, in order to provide theoretical basis and technical support for crude oil pollution bioremediation. The main contents and results were summarized as follows: 1. A highly efficient crude oil-degrading bacterium of D. sp. CN-3 was isolated from the marine sediments of Penglai 19-3 platform in the Bohai Sea. The strain degraded a wide variety of petroleum hydrocarbons, alkanes and aromatic hydrocarbons, including linear alkanes (C10-C36), branched alkanes (pristane and phytane), cycloalkanes and aromatic hydrocarbons (phenanthrene and pyrene). Besides, the strain had good tolerance to different temperature (4-42℃), pH (5-10) and salinity (0-160 g/L). Particularly, the petroleum hydrocarbon degradation activity was hardly influenced under high salinity conditions (85 g/L). In order to acceletate the petroleum hydrocarbons degradation, D. sp. CN-3 was able to produce surfactants or change cell surface hydrophobicity. 2. The complete genome sequence of D. sp. CN-3 was achieved, providing a comprehensive genetic background for different biological processes and metabolic pathways investigations. The genome contained a chromosome genome and two plasmid genomes, with total length of 3741379 bp and GC content of 70.66%. There were 3496 encoding genes, whose length accounted for 88.83% of the genome. The genome contained 50 tRNA, 9 rRNA and 12 gene islands. Large numbers of genes associated with alkane degradation were annotated, including one alkane hydroxylase gene alkB and one CYP153 of cytochrome P450 family, as well as variety of enzymes involved in alkane single-terminal oxidation pathway, such as alcohol dehydrogenase, aldehyde dehydrogenase and acyl-CoA synthetase, etc. In addition, 19 genes related to the aromatic compounds degradation were annotated in the genome, such as catechol 1,2-dioxygenase gene (catA), catechol 2,3-dioxygenase gene (catE), etc. It was speculated that the catechol pathway played a major role in aromatic compounds degradation by D. sp. CN-3, due to relatively complete genes in the genome. 3. Transcriptome sequencing of D. sp. CN-3 cultivated in sodium succinate and n-hexadecane cultures was completed to comprehensively analyze the genes expression in different biological processes. Transcriptome analysis indicated that the transcription profiles of CN-3 were significantly changed under n-hexadecane induction, and 546 differentially expressed genes (FDR<0.05 and |Log2FC|>1) were found, among which 168 genes were up-regulated and 378 genes were down-regulated. Many differentially expressed genes were related to lipid metabolism, amino acid metabolism, carbohydrate metabolism, xenobiotics biodegradation and metabolism, as well as transcription and translation. Potential new genes (31) were also found herein. In addition, alkB, CYP153, Fdx and FdR were strongly up-regulated (log2FC 5.3065-6.198), and genes related to the downstream metabolism of n-hexadecane were also up-regulated in different degrees, which could be speculated that CN-3 strain degraded n-hexadecane through the single-terminal oxidation pathway. 4. Taking advantage of in vitro and in vivo experiments, the molecular mechanism of alkB and CYP153 in division and cooperation of alkane degradation had been confirmed, which was considered as a potential new mechanism and quite different from previous reports. Through amplification and homology analysis of alkB and CYP153, it was found that there were four conserved motifs of Hist-1, Hist-2, Hist-3 and HYG-motif in the amino acid sequences, which were identified as AlkB alkane hydroxylase, and the CYP153 gene belonged to the CYP153A family of cytochrome P450. Using suicide plasmid of pK18, the gene disruption mutants of ΔalkB and ΔCYP were successfully constructed. It was confirmed that both alkB and CYP153 played important roles in the middle-chain alkanes, branched alkanes and long-chain alkanes degradation, but they had their own preferences. Specifically, CYP153 was mainly responsible for degradation of middle-chain alkanes and branched alkanes, and alkB was mainly responsible for long-chain alkanes degradation. There was a synergistic effect between alkB and CYP153 genes in C28 degradation. Meanwhile, these results were consistent with the transcriptional level analysis of alkB and CYP153 genes induced by different alkanes. From the perspective of synthetic biology, Pseudomonas putida F1 was used as the platform cell to introduce alkB gene to construct petroleum hydrocarbon-degrading cell factory. Consequently, P. putida F1 could degrade medium-chain and long-chain alkane (C14, C16, C24 and C26) and be potential in crude oil pollution bioremediation. 5. Relying on different petroleum degradation characteristics and biosurfactant production abilities, D. sp. CN-3 and Acinetobacter sp. HC8-3S, were functionally combined to construct a bacterial consortium. Compared to single strains, the petroleum degradation of this consortium was significantly enhanced, with 95.8% of degradation efficiency in 10 days, which degraded petroleum efficiently in the ranges of pH (4-10) and salinity (0-120 g/L). In the crude oil-contaminated soil microcosms, the degradation rate of bioaugmented treatment with the consortium was 485.8 mg kg-1 d-1, which was significantly higher than many previous reports, demonstrating the application potential of the consortium in crude oil pollution bioremediation. In a summary, the petroleum hydrocarbon degradation performance and mechanisms, as well as alkane hydroxylase function in D. sp. CN-3 were investigated herein. The molecular mechanism of alkB and CYP153 in division and cooperation of alkane degradation had been confirmed to be a potential new one. This is valuable to provide references for mechanism research and molecular modification in other alkane-degrading strains, and provide theoretical basis and technical support for bioremediation of crude oil pollution.
MOST Discipline Catalogue理学::海洋科学
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/25308
Collection中科院烟台海岸带研究所知识产出
Recommended Citation
GB/T 7714
陈卫卫. 迪茨氏菌CN-3的石油烃降解机制及烷烃羟化酶功能研究[D]. 北京. 中国科学院大学,2020.
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